The present invention relates to an apparatus and method for verifying the drop delay in a flow cytometer. More particularly, the present invention relates to an apparatus and method which detects the presence or absence of particles of interest in fluid droplets formed by a flow cytometer to determine whether the time at which the fluid droplets are being charged is correct, so that the droplets can be sorted electrostatically with precision.
Flow cytometers known in the art are used for analyzing and sorting particles in a fluid sample, such as cells of a blood sample or particles of interest in any other type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing a sheath fluid. The flow cytometer transports the particles (hereinafter called xe2x80x9ccellsxe2x80x9d) in the fluid sample as a cell stream to a flow cell, while also directing the sheath fluid to the flow cell.
Within the flow cell, a liquid sheath is formed around the cell stream to impart a substantially uniform velocity on the cell stream. The flow cell hydrodynamically focuses the cells within the stream to pass through the center of a laser beam. The point at which the cells intersect the laser beam, commonly known as the interrogation point, can be inside or outside the flow cell. As a cell moves through the interrogation point, it causes the laser light to scatter. The laser light also excites components in the cell stream that have fluorescent properties, such as fluorescent markers that have been added to the fluid sample and adhered to certain cells of interest, or fluorescent beads mixed into the stream.
The flow cytometer further includes an appropriate detection system consisting of photomultiplier tubes, photodiodes or other light detecting devices, which are focused at the intersection point. The flow cytometer analyzes the detected light to measure physical and fluorescent properties of the cell. The flow cytometer can further sort the cells based on these measured properties.
To sort cells by an electrostatic method, the desired cell must be contained within an electrically charged droplet. To produce droplets, the flow cell is rapidly vibrated by an acoustic device, such as a piezoelectric element. The droplets form after the cell stream exits the flow cell and at a distance downstream from the interrogation point. Hence, a time delay exists from when the cell is at the interrogation point until the cell reaches the actual break-off point of the droplet. The magnitude of the time delay is a function of the manner in which the flow cell is vibrated to produce the droplets, and generally can be manually adjusted, if necessary.
To charge the droplet, the flow cell includes a charging element whose electrical potential can be rapidly changed. Due to the time delay which occurs while the cell travels from the interrogation point to the droplet break-off point, the flow cytometer must invoke a delay period between when the cell is detected to when the electrical potential is applied to the charging element. This charging delay is commonly referred to as the xe2x80x9cdrop delayxe2x80x9d, and should coincide with the travel time delay for the cell between the interrogation point and the droplet break-off point to insure that the cell of interest is in the droplet being charged.
At the instant the desired cell is in the droplet just breaking away from the cell stream, the charging element is brought up to the appropriate potential, thereby causing the droplet to isolate the charge once it is broken off from the stream. The electrostatic potential from the charging circuit cycles between different potentials to appropriately charge each droplet as it is broken off from the cell stream.
Because the cell stream exits the flow cell in a substantially downward vertical direction, the droplets also propagate in that direction after they are formed. To sort the charged droplet containing the desired cell, the flow cytometer includes two or more deflection plates held at a constant electrical potential difference. The deflection plates form an electrostatic field which deflects the trajectory of charged droplets from that of uncharged droplets as they pass through the electrostatic field. Positively charged droplets are attracted by the negative plate and repelled by the positive plate, while negatively charged droplets are attracted to the positive plate and repelled by the negative plate. The lengths of the deflection plates are small enough so that the droplets which are traveling at high velocity clear the electrostatic field before striking the plates. Accordingly, the droplets and the cells contained therein can be collected in appropriate collection vessels downstream of the plates.
Known flow cytometers similar to the type described above are described, for example, in U.S. Pat. Nos. 3,960,449, 4,347,935, 4,667,830, 5,464,581, 5,483,469, 5,602,039, 5,643,796 and 5,700,692, the entire contents of each patent being incorporated by reference herein. Other types of known flow cytometer, are the FACSVantage(trademark), FACSort(trademark), FACSCount(trademark), FACScan(trademark) and FACSCalibur(trademark) systems, each manufactured by Becton Dickinson and Company, the assignee of the present invention.
As can be appreciated from the foreign description, in order for a flow cytometer to correctly sort cells of interest, the drop delay must be precisely measured to ensure that a cell of interest which was detected at the interrogation point is actually present in the droplet being charged. If the drop delay is not accurately determined, it is likely that the charge will be applied to a droplet formed earlier or later than the droplet containing the cell of interest. In this event, the droplet containing the cell of interest will not be charged, and therefore will not be sorted as desired. Rather, an incorrectly charged droplet will be sorted, thus reducing the overall sorted cell count, or adding an unwanted cell to the cell count if that droplet contains an unwanted cell.
Several known methods exist for calculating the drop delay with reasonable accuracy. In one known method, the distance between the interrogation point and the droplet formation (break-off) point is measured using, for example, a graduated optical measuring tool. The measuring tool is then repositioned so that the graduation originally positioned at the interrogation point is moved to the droplet break-off point, and the graduation originally positioned at the droplet break-off point is positioned in the droplet stream. The number of droplets appearing between the graduation positioned at the droplet break-off point and the graduation in the droplet stream is then counted, and the drop delay is expressed as the number of counted drops.
For example, if the number of counted drops appearing between the graduations is equal to five, this indicates that five drop periods elapse form the time a cell is at the interrogation point until it reaches the droplet break-off point. Accordingly, the charge timing of the flow cytometer is set so that charging intended to be applied to a droplet containing a cell of interest is delayed by five drop periods from the time when the cell of interest is detected at the interrogation point.
Although this method generally enables the flow cytometer to charge the correct droplets, and therefore sort the cells of interest with reasonable accuracy, the method provides no means to verify the accuracy of the charge timing while cell sorting is being performed. Rather, the results of the sort must be examined after all or at least a portion of the sorting process has been completed. If, upon examination of the results, it is determined that the charge timing was incorrect, the process must be repeated until the correct charge timing is determined. Furthermore, because the method requires multiple steps, such as aligning the graduations on the optical instrument with the appropriate points at different positions along the droplet stream, the process can be somewhat time consuming even if the charge timing is correctly set on the initial attempt.
Instead of attempting to estimate the drop delay as in the method described above, another known method exists in which a number of sort results are obtained for a calibration sample sorted at different droplet charge timing settings, and the sort results are empirically checked. That is, before attempting to sort an actual cell sample, the sample reservoir is filled with sample particles, commonly known as xe2x80x9cbeadsxe2x80x9d, which have physical and fluorescent characteristics similar to those of the cells of interest in the actual sample. The flow cytometer is run for a time period during which a predetermined number of beads (for example, 40 beads) should be sorted, with the drop delay for charging being set at a first estimated setting. As the cells are being sorted, they are collected on an area of a collection medium, such as a glass slide suitable for viewing under a microscope.
The sorting is then performed again for the same amount of time with the drop delay for charging being set at a slightly different value, and the sorted cells are collected on a different area of the collection medium. An additional number of sorts are performed, with the drop delay being set differently for each sort, and with the results of each sort being collected at a different region on the collection medium. After the desired number of sorts have been performed, each sorted sample on the collection medium is viewed under a microscope, and the number of beads actually sorted for each sorted operation is counted. The drop delay yielding the highest number of sorted cells is then determined to be the most appropriate drop delay at which the actual cell samples should be sorted.
Although this method enables the drop delay to be set with reasonable accuracy, the method requires that multiple sorts be performed on the sample beads, and thus is fairly time consuming. The sample collection medium must be removed from the flow cytometer and examined under a microscope to analyze the samples, thus making the process more complicated and subject to error. Also, because the number of cells in each of the collected samples must be counted manually, the time required to complete the analysis is further increased, and additional opportunity for error is introduced. In addition, the method provides no means to verify the accuracy of the charge timing while the sorting operation is being performed
Accordingly, a need exist for an efficient and reliable apparatus and method for detecting the drop delay in a flow cytometer, so that the drop delay can be accurately set and maintained to insure reliable cell sorting.
An object of the present invention is to provide an accurate and reliable apparatus and method for verifying the accuracy of the drop delay in a flow cytometer, so that flow cytometer can accurately charge droplets containing particles of interest to insure that the particles of interest are sorted with high reliability.
Another object of the invention is to provide an apparatus and method for setting the drop delay at which droplets are charged while the flow cytometer is performing the process of sampling and sorting particles of interest.
A further object of the invention is to provide an apparatus and method which analyzes the contents of droplets formed in a flow cytometer to determine whether the droplets are being charged at the appropriate drop delay so that droplets containing particles of interest are sorted in the appropriate manner.
These and other object of the present invention are substantially achieved by providing an apparatus and method for verifying the drop delay in a flow cytometer. The drop delay is defined as the period that elapses from the moment at which a particle of interest is detected at an interrogation point in the flow cytometer to the moment at which a charging device applies a charging potential to the droplet forming at the droplet break-off point. Preferably, the drop delay should coincide with the amount of time that elapses for the particle of interest to travel from the interrogation point to a location that substantially coincides with the droplet break-off point. The apparatus and method analyzes the content of the droplets formed by the flow cytometer, and based on the analysis, determines the drop delay at which the flow cytometer should be operating. The content of the droplets can be analyzed to detect for the presence or absence of a particle of interest that was detected at the interrogation point in the flow cytometer. The drop delay at which the droplets being formed by the flow cytometer are charged can then be set based on the results of the analysis of the contents of the droplets, to ensure that droplets containing particles of interest are charged and thus sorted by the flow cytometer, while droplets not including particles of interest remain uncharged and are not sorted.
The apparatus and method preferably employs non-contact sensors, such as light detectors or the like, for analyzing the content of the droplets. The content of the droplets can be analyzed at the droplet break-off point at which the droplets are initially formed, or at any location in the uncharged or charged droplet streams.